Further Errors in the Acetylene Reduction Assay: Effects of Plant Disturbance

Abstract

Minchin, F. R., Sheehy, J. E. and Witty, J. F. 1986. Further errors in the acetylene reduction assay: effects of plant disturbance.—J. exp. Bot. 37: 1581-1591. A flow-through gas system was used to study the effects of disturbance on nitrogenase (acetylene reduction) activity of nodulated root systems of soyabean (Glycine max) and white clover (Trifolium repens). Detopping plus removal of the rooting medium (by shaking) produced a substantial decrease in maximum nitrogenase activity. This response is due to a reduction in oxygen flux to the bacteroids caused by an increase in the oxygen diffusion resistance of the nodule. The decrease in maximum nitrogenase activity was much smaller for roots subjected to detopping only. Thus, the effect of root shaking is more important than that of shoot removal. The effect of detopping plus root shaking on nitrogenase activity occurred whether the plants were equilibrated and assayed at 25 °C or 15 °C. However, the effect of disturbance on the oxygen diffusion resistance of the nodules, and thus on nitrogenase activity, was greater at the higher temperature. At the lower temperature the oxygen diffusion resistance of the nodules had already been increased in response to the reduced requirement for oxygen. These nodules were less susceptible to the effects of disturbance. Thus, comparisons of the effects of equilibration temperature on nitrogenase activity produced different results depending on whether intact or disturbed systems were used. With intact systems activity was lower at the lower temperature but with detopped/shaken roots the lowest activity occurred at the higher temperature. It is concluded that the use of detopped/shaken roots can produce substantial errors in the acetylene reduction assay, which makes the assay invalid even when used for comparative purposes. However, comparisons with rates of 15N2 fixation and H2 production show that accurate measurements of nitrogenase activity can be obtained from maximum rates of acetylene reduction by intact plants in a flow-through gas system. The continued use of assay procedures in which cumulated